Investigation of the biosynthesis of the pipecolate moiety of neuroprotective polyketide meridamycin

Jiang, Hao; Haltli, Brad; Feng, Xidong; Cai, Ping; Summers, Mia Y.; Lotvin, Jason; He, Min

doi:10.1038/ja.2011.45

Cited by 10 publications

(7 citation statements)

References 24 publications

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“…362,363 Enzymatic oxidation at the C 2 amine yields the acyclic imine 498 and NADH. Capture by the C 6 amine gives 499 , and loss of NH 3 from the tetrahedral adduct yields the Δ 1 -cyclic imine 500 .…”

Section: Nonradical Cyclization Mechanismsmentioning

confidence: 99%

“…The enzymatic conversion of the proteinogenic amino acid L-lysine (497) to the six-member heterocyclic pipecolic acid (501) is a simple example of this ring-forming logic (Scheme 71). 362,363 Enzymatic oxidation at the C 2 amine yields the acyclic imine 498 and NADH. Capture by the C 6 amine gives 499, and loss of NH 3 from the tetrahedral adduct yields the Δ 1 -cyclic imine 500.…”

Section: Capture Of Transient Carbonylsmentioning

confidence: 99%

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Oxidative Cyclization in Natural Product Biosynthesis

et al. 2016

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Oxidative cyclizations are important transformations that occur widely during natural product biosynthesis. The transformations from acyclic precursors to cyclized products can afford morphed scaffolds, structural rigidity and biological activities. Some of the most dramatic structural alterations in natural product biosynthesis occur through oxidative cyclization. In this review, we examine the different strategies used by Nature to create new intra-(inter-)-molecular bonds via redox chemistry. The review will cover both oxidation- and reduction-enabled cyclization mechanisms, with an emphasis on the former. Radical cyclizations catalyzed by P450, nonheme iron, α-KG dependent oxygenases and radical SAM enzymes are discussed to illustrate the use of molecular oxygen and S-adenosylmethionine to forge new bonds at unactivated sites via one-electron manifolds. Nonradical cyclizations catalyzed by flavin-dependent monooxygenases and NAD(P)H-dependent reductases are covered to show the use of two-electron manifolds in initiating cyclization reactions. The oxidative installation of epoxides and halogens into acyclic scaffolds to drive subsequent cyclizations are separately discussed as examples of “disappearing” reactive handles. Lastly, oxidative rearrangement of rings systems, including contractions and expansions will be covered.

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Section: Nonradical Cyclization Mechanismsmentioning

confidence: 99%

Section: Capture Of Transient Carbonylsmentioning

confidence: 99%

Oxidative Cyclization in Natural Product Biosynthesis

et al. 2016

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“…It closes the ring structure in a final cyclization step (Motamedi et al, 1997;Motamedi & Shafiee, 1998;Goranovic et al, 2010;Andexer et al, 2011;Mo et al, 2011) before the immature macrolactone is further processed by post-PKS tailoring enzymes resulting in the final compound FK506 (Motamedi et al, 1996;Chen et al, 2013) (Figure 1). Pipecolic acid, a non-proteinogenic amino acid, is a key intermediate in the synthesis of a large number of drugs, e.g., pristinamycin (Mast et al, 2011), friulimicin (Müller et al, 2007), meridamycin (Jiang et al, 2011), rapamycin (Gatto et al, 2006), tacrolimus (Turlo et al, 2012), nocardiospin (Bis et al, 2015), tubulysin B (Steinmetz et al, 2004) and others. In fact, pipecolic acid is often even crucial for the bioactivity of secondary metabolites (Min, 2018).…”

Section: Introductionmentioning

confidence: 99%

Optimization of the precursor supply for an enhanced FK506 production in Streptomyces tsukubaensis

Schulz

Schall

Stehle

et al. 2022

Front. Bioeng. Biotechnol.

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Tacrolimus (FK506) is a macrolide widely used as immunosuppressant to prevent transplant rejection. Synthetic production of FK506 is not efficient and costly, whereas the biosynthesis of FK506 is complex and the level produced by the wild type strain, Streptomyces tsukubaensis, is very low. We therefore engineered FK506 biosynthesis and the supply of the precursor L-lysine to generate strains with improved FK506 yield. To increase FK506 production, first the intracellular supply of the essential precursor lysine was improved in the native host S. tsukubaensis NRRL 18488 by engineering the lysine biosynthetic pathway. Therefore, a feedback deregulated aspartate kinase AskSt* of S. tsukubaensis was generated by site directed mutagenesis. Whereas overexpression of AskSt* resulted only in a 17% increase in FK506 yield, heterologous overexpression of a feedback deregulated AskCg* from Corynebacterium glutamicum was proven to be more efficient. Combined overexpression of AskCg* and DapASt, showed a strong enhancement of the intracellular lysine pool following increase in the yield by approximately 73% compared to the wild type. Lysine is coverted into the FK506 building block pipecolate by the lysine cyclodeaminase FkbL. Construction of a ∆fkbL mutant led to a complete abolishment of the FK506 production, confirming the indispensability of this enzyme for FK506 production. Chemical complementation of the ∆fkbL mutant by feeding pipecolic acid and genetic complementation with fkbL as well as with other lysine cyclodeaminase genes (pipAf, pipASt, originating from Actinoplanes friuliensis and Streptomyces pristinaespiralis, respectively) completely restored FK506 production. Subsequently, FK506 production was enchanced by heterologous overexpression of PipAf and PipASp in S. tsukubaensis. This resulted in a yield increase by 65% compared to the WT in the presence of PipAf from A. friuliensis. For further rational yield improvement, the crystal structure of PipAf from A. friuliensis was determined at 1.3 Å resolution with the cofactor NADH bound and at 1.4 Å with its substrate lysine. Based on the structure the Ile91 residue was replaced by Val91 in PipAf, which resulted in an overall increase of FK506 production by approx. 100% compared to the WT.

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“…Pipecolic acid, a non-proteinogenic amino acid, is a key intermediate in the synthesis of a large number of drugs, e.g. pristinamycin (Mast et al, 2011), friulimicin (Müller et al, 2007), meridamycin (Jiang et al, 2011), rapamycin (Gatto et al, 2006), tacrolimus (Turlo et al, 2012), nocardiospin (Bis et al, 2015), tubulysin B (Steinmetz et al, 2004) and others. In fact, pipecolic acid is often even crucial for the bioactivity of secondary metabolites (Min, 2006).…”

Section: Introductionmentioning

confidence: 99%

Optimization of the precursor supply for an enhanced FK-506 production in Streptomyces tsukubaensis

Schulz

Schall

Stehle

et al. 2022

Preprint

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Tacrolimus (FK-506) is a macrolide widely used as immunosuppressant to prevent transplant rejection. Synthetic production of FK-506 is not efficient and costly, whereas the biosynthesis of FK-506 is complex and the level produced by the wild type strain, Streptomyces tsukubaensis, is very low. We therefore engineered FK-506 biosynthesis and the supply of the precursor L-lysine to generate strains with improved FK-506 yield. To increase FK-506 production, first the intracellular supply of the essential precursor lysine was improved in the native host S. tsukubaensis by engineering the lysine biosynthetic pathway. Therefore, a feedback deregulated aspartate kinase AskSt* of S. tsukubaensis was generated by site directed mutagenesis. Whereas overexpression of AskSt* resulted only in a 17% increase in FK-506 yield, heterologous overexpression of a feedback deregulated AskCg* from Corynebacterium glutamicum was proven to be more efficient. Combined overexpression of AskCg* and DapASt, showed a strong enhancement of the intracellular lysine pool following increase in the yield by approximately 73% compared to the wild type. Lysine is coverted into the FK-506 building block pipecolate by the lysine cyclodeaminase FkbL. Construction of a ∆fkbL mutant led to a complete abolishment of the FK-506 production, confirming the indispensability of this enzyme for FK-506 production. Chemical complementation of the ∆fkbL mutant by feeding pipecolic acid and genetic complementation with fkbL as well as with other lysine cyclodeaminase genes (pipAf, pipASt, originating from Actinoplanes friuliensis and Streptomyces pristinaespiralis, respectively) completely restored FK-506 production. Subsequently, FK-506 production was enchanced by heterologous overexpression of PipAf and PipASp in S. tsukubaensis. This resulted in a yield increase by 65% compared to the WT in the presence of PipAf from A. friuliensis. For further rational yield improvement, the crystal structure of PipAf from A. friuliensis was determined at 1.3 Å resolution with the cofactor NADH bound and at 1.4 Å with its substrate lysine. Based on the structure the Ile91 residue was replaced by Val91 in PipAf, which resulted in an overall increase of FK-506 production by approx. 100% compared to the WT.

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Investigation of the biosynthesis of the pipecolate moiety of neuroprotective polyketide meridamycin

Cited by 10 publications

References 24 publications

Oxidative Cyclization in Natural Product Biosynthesis

Oxidative Cyclization in Natural Product Biosynthesis

Optimization of the precursor supply for an enhanced FK506 production in Streptomyces tsukubaensis

Optimization of the precursor supply for an enhanced FK-506 production in Streptomyces tsukubaensis

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